RESEARCH Open Access

HMGB1 and thrombin mediate theblood-brain barrier dysfunction actingas biomarkers of neuroinflammationand progression to neurodegenerationin Alzheimer’s diseaseBarry W. Festoff1,2, Ravi K. Sajja3, Patrick van Dreden4 and Luca Cucullo3*

Abstract

Background: The blood-brain barrier (BBB) dysfunction represents an early feature of Alzheimer’s disease (AD) thatprecedes the hallmarks of amyloid beta (amyloid β) plaque deposition and neuronal neurofibrillary tangle (NFT)formation. A damaged BBB correlates directly with neuroinflammation involving microglial activation and reactiveastrogliosis, which is associated with increased expression and/or release of high-mobility group box protein 1(HMGB1) and thrombin. However, the link between the presence of these molecules, BBB damage, and progression toneurodegeneration in AD is still elusive. Therefore, we aimed to profile and validate non-invasive clinical biomarkers ofBBB dysfunction and neuroinflammation to assess the progression to neurodegeneration in mild cognitive impairment(MCI) and AD patients.

Methods: We determined the serum levels of various proinflammatory damage-associated molecules in aged controlsubjects and patients with MCI or AD using validated ELISA kits. We then assessed the specific and direct effects ofsuch molecules on BBB integrity in vitro using human primary brain microvascular endothelial cells or a cell line.

Results: We observed a significant increase in serum HMGB1 and soluble receptor for advanced glycation endproducts (sRAGE) that correlated well with amyloid beta levels in AD patients (vs. control subjects). Interestingly, serumHMGB1 levels were significantly elevated in MCI patients compared to controls or AD patients. In addition, as a markerof BBB damage, soluble thrombomodulin (sTM) antigen, and activity were significantly (and distinctly) increased in MCIand AD patients. Direct in vitro BBB integrity assessment further revealed a significant and concentration-dependentincrease in paracellular permeability to dextrans by HMGB1 or α-thrombin, possibly through disruption of zonaoccludins-1 bands. Pre-treatment with anti-HMGB1 monoclonal antibody blocked HMGB1 effects and leaving BBBintegrity intact.

Conclusions: Our current studies indicate that thrombin and HMGB1 are causal proximate proinflammatory mediatorsof BBB dysfunction, while sTM levels may indicate BBB endothelial damage; HMGB1 and sRAGE might serve as clinicalbiomarkers for progression and/or therapeutic efficacy along the AD spectrum.

Keywords: Biomarkers, Blood-brain barrier, Clinical, DAMPS, HMGB1, Neuroinflammation, Neurodegeneration,Permeability, Thrombin

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* Correspondence: luca.cucullo@ttuhsc.edu3Department of Pharmaceutical Sciences, Texas Tech University HealthSciences Center, 1300 S. Coulter Street, Amarillo, TX 79106, USAFull list of author information is available at the end of the article

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Festoff et al. Journal of Neuroinflammation (2016) 13:194 DOI 10.1186/s12974-016-0670-z

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Abbreviations: ANOVA, Analysis of variance; AD, Alzheimer’s disease; Aβ, Amyloid beta; BBB, Blood-brain barrier;DAMPs, Damage-associated molecular patterns; EC, Endothelial cell; ELISA, Enzyme-linked immunosorbent assay;hCMEC/D3, Human cerebromicrovascular endothelial cell line; HMGB1, High-mobility group box protein 1;HBMEC, Primary human brain microvascular endothelial cells; IL-1β, Interleukin-1 beta; MCI, Mild cognitive impairment;NFT, Neurofibrillary tangles; NVU, Neurovascular unit; PAR, Protease-activated receptor; RITC, Rhodamine Bisothiocyanate; sRAGE, Soluble receptor for advanced glycation end products; TJ, Tight junction; TLR, Toll-like receptor;TM, Thrombomodulin; TNF, Tumor necrosis factor; WBCs, White blood cells; ZO-1, Zona occludins

BackgroundDespite overarching evidence, the amyloid hypothesisin Alzheimer’s disease (AD) first elaborated in 1991,has yet to provide positive outcomes notwithstandingthe billions spent on clinical trials [1]. The neuropatho-logical hallmarks of AD are extracellular amyloid beta(Aβ)/neuritic plaques and intracellular neurofibrillarytangle (NFT) formation [2]. In association with thesehallmarks, soluble Aβ levels increase in the blood, bothin AD patients and transgenic mouse models [3–5],while the brain amyloid β aggregates promote a neu-roinflammatory response mediated by activated micro-glia, astrocytes, and microvascular endothelial cells(ECs) [6, 7]. Since late-onset AD (LOAD) is not associ-ated with such manifestations information from AD,transgenic animal models cannot be fully extrapolatedto human AD pathology. Furthermore, microglial acti-vation and other aspects of neuroinflammation involv-ing oxidative stress (reactive oxygen species (ROS),nitric oxide (NO)) actually precede neuronal damage[8, 9], prior to AD histopathologic lesions. Moreover, acritical characteristic of neuroinflammation is the dis-ruption of the blood-brain barrier (BBB) that extendsbeyond the tissue or cellular pathophysiology of endo-thelial cell (EC) dysfunction to the neurovascular unit(NVU), including astrocytic end-feet, microglia, neu-rons, and pericytes [10–12]. Recent National Institutesof Health (NIH) workshops have emphasized key areasthat must be focused on as it relates to AD neuroin-flammation research involving the BBB: (1) transport ofAβ and other macromolecules in and out of the brain,i.e., permeability; (2) BBB as both the source and targetof inflammatory factors; and (3) effects of oxidativestress, ROS, and NO on BBB. Besides astrogliosis, acti-vation and transmigration of blood-borne substancesand circulating immune cells into the CNS is a less stud-ied and underappreciated area in AD research [13–16].The precise molecular factors governing the initial BBBdamage leading to neurodegeneration, in general, and AD,in particular, are not well understood.Thrombin and high-mobility group box protein 1

(HMGB1) are key molecules of two most potent hostdefense systems that converge on the innate immunesystem, coagulation, and inflammation. We postulated

that they may play significant roles in the BBB disruptionsince both are proinflammatory and both are known todisrupt vascular barriers in other tissues [17–20]. Throm-bin is a proinflammatory serine protease that is wellknown for its essential role as the ultimate protease in thecoagulation pathway. HMGB1 is a non-histone nuclearprotein with dual functions depending on localization.Within the cells, it is localized primarily to the nucleuswhere it binds DNA and plays a role in transcriptionalregulation [21]. However, extracellular HMGB1 serves asa proinflammatory cytokine and is a late mediator ofsepsis [22]. Beyond infections, HMGB1 has pathogenicroles during trauma and sterile inflammation, such assystemic inflammatory response syndrome (SIRS), whereelevated levels in sera orchestrate key events includingleukocyte recruitment and white blood cell (WBC) induc-tion to secrete inflammatory cytokines [23, 24].Relevant to our studies, HMGB1 impairs memory

behavior in mice that is mediated via Toll-like receptor 4(TLR4) and the receptor for advanced end product gly-cation (RAGE) [25]. These pre-clinical data correlatewith clinical studies showing that sepsis survivors havepermanent cognitive deficits [26] and that these mayalso be mediated via HMGB1, but the precise mechan-ism remains unknown. HMGB1 and another alarmin,S100B, along with Aβ, are now considered as three sig-nificant damage or danger-associated molecular patterns(DAMPs) that “fan the flame” [27] of neuroinflammationin AD [28]. How they might do this is currently unknown.As an approach to this problem, we first measured levelsof these DAMPs in mild cognitive impairment (MCI), AD,and normal aged subjects and then used pure proteins inthe range of these levels to perturb human BBB functionin vitro.

MethodsHuman subjects and specimen collectionThe human study was approved by the institutionalreview board at the University of Kansas Medical Center(KUMC) and was performed in compliance with theHelsinki Declaration. All subjects were recruited fromthe KU Alzheimer Disease Center (ADC) following awritten informed consent, and all demographic informa-tion was de-identified. Subjects in the AD, MCI, and

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aged control cohorts met the criteria as outlined in the2011–2012 NIA/Alzheimer’s Association Guidelines forAD by McKhann et al. [29] and for MCI by Albert et al.[30]. Furthermore, all subjects were screened by clinicaldementia rating (CDR) and neuropsychological testingas defined by the Uniform Data Set of the AlzheimerDisease Centers and underwent a consensus review.MCI patients were further classified as “MCI due to AD”if another more likely cause was not identified. Our MCIcohort patients were all designated MCI due to AD.Venous blood collection was performed with BD

Vaccutainer® blood collection tubes and centrifuged. Theserum samples were aliquoted and stored at −80 °C untilanalysis. Demographic information for these patientgroups is shown in Table 1.

Cell cultureThe human cerebromicrovascular endothelial cell line(hCMEC/D3) was a gift from Dr. P.O. Couraud(INSERM, France). This cell line has been widely usedas a representative model of human BBB in vitro formechanistic studies involving the molecular regulationof BBB integrity [31]. Cells between passages 28 and 31were cultured in HEPES-buffered EBM-2 media supple-mented with growth factors and antibiotics and main-tained at 37 °C with 5 % CO2 exposure. In preliminaryexperiments, primary human brain microvascular endo-thelial cells (HBMEC) were cultured in Dr. KY Kim’slaboratory (KUMC) as described previously [32].

Analysis of serum biomarkersHuman serum samples were thawed on ice and analyzedby enzyme-linked immunosorbent assay (ELISA) forquantification of HMGB1 (Novatein Biosciences, Woburn,MA), s100β (EMD Millipore, Billerica, MA), amyloid β(aa 1-42), and soluble receptor for advanced glycationend products (sRAGE) from R&D Systems (Minneapolis,MN, USA), according to the manufacturers’ instructions.Soluble thrombomodulin (sTM) antigen (TMa) levelswere assayed by kit (Stago, Gennevilliers, France), andsTM activity was measured as described previously [33].

BBB integrity assessmentCells were seeded on Corning Transwell® polyestermembrane inserts (12-well, 0.4 μm pore size) as previ-ously described [31]. Monolayer integrity and morph-ology were periodically assessed by transendothelialelectrical resistance (TEER; Ω cm2) and phase contrastmicroscopy, respectively. Cells were maintained in treat-ment media (EMB-2 supplemented with 10 mM HEPES,antibiotics, and 1 % human serum) overnight prior totreatment with human recombinant HMGB1 protein(rHMGB1) with endotoxin levels lower than 0.1 EU permicrogram of the protein (R&D Systems, Minneapolis,

MN, USA). Following 3 h of treatment with rHMGB1(5, 10, and 50 ng/mL), α-thrombin (0.5, 1, and 5 nM) orcontrol media, inserts were transferred to new 12-wellplates and transport buffer added (pre-warmed HBSSbuffer with antibiotics and 0.5 % BSA) to the apical(500 μL) and basal compartment (1000 μL). After incu-bation for 20 min at 37 °C under 5 % CO2 exposure, a

Table 1 Human subject demographics (gender and age wereshown for each subject in AD, MCI, and control cohorts at thetime of blood collection)

Patient ID Gender Age at draw Dx at draw

1 F 80 AD

2 F 77 AD

3 F 81 AD

4 F 60 AD

5 M 63 AD

6 M 76 AD

7 M 73 AD

8 M 81 AD

9 M 67 AD

10 M 70 AD

11 M 84 AD

12 M 88 AD

1 F 67 MCI

2 M 88 MCI

3 M 76 MCI

4 M 74 MCI

5 M 77 MCI

6 M 64 MCI

7 M 72 MCI

8 M 84 MCI

9 M 68 MCI

10 M 72 MCI

11 M 75 MCI

12 M 65 MCI

1 F 77 NL

2 F 76 NL

3 F 78 NL

4 F 66 NL

5 F 69 NL

6 F 74 NL

7 F 81 NL

8 F 75 NL

9 F 72 NL

10 F 66 NL

11 M 69 NL

12 M 76 NL

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mixture of florescent dextrans of increasing molecularsizes (FITC-4 kDa, 10 mg/mL; Cascade Blue 10 kDa,5 mg/mL; and rhodamine B isothiocyanate (RITC)-70 kDa, 10 mg/mL) was added to the apical compartment.Dextran permeability (luminal to abluminal flux) wasmeasured at 30 min following the addition of dextrans.Apparent permeability coefficients (Pe; centimeters persecond) were calculated and expressed as percent control[31]. For pharmacological inhibition (validation) studies,cells were co-incubated with anti-HMGB1 antibody (giftfrom Dr. Michael Bustin, NIH) or non-specific controlantibody for the indicated duration.

Immunofluorescence analysisImmunocytochemistry (ICH) for zona occludin-1(ZO-1) was performed using primary anti-ZO-1 anti-body (Zymed Laboratories, San Francisco, CA), AlexaFluor 488-conjugated secondary antibody and 4,6-dia-midino-2-phenylindole (DAPI; Molecular Probes, Eu-gene, OR) as described in [32].

Statistical analysisData were expressed as mean ± SEM or SD. Data wereanalyzed by one-way analysis of variance (ANOVA)followed by Dunnett’s or Tukey’s post hoc multiplecomparison tests using GraphPad Prism Software Inc.(La Jolla, CA, USA). P value was set to less than 0.05 forstatistical significance.

ResultsSerum profile of proinflammatory “damage-associated”factors in AD, MCI, and age-matched normal subjectsPart of the criteria for AD and MCI due to AD infersthat other more likely diagnoses are less likely than AD.Although it does not totally eliminate, it clearly shouldlimit contamination by other diagnoses such as vasculardementia, Parkinson’s disease with dementia, and pri-mary systemic inflammatory diseases.Levels of various proinflammatory markers including

Aβ, sRAGE, HMGB1, and S100β in serum samples de-rived from control subjects (NL), AD, or MCI patients(see Table 1 for demographic information) were mea-sured by ELISA. As shown in Fig. 1, Aβ levels weresignificantly higher in AD patients when compared toMCI and NL. No statistically significant differences werenoted between MCI and NL groups. An unprecedentedpathophysiological parallelism between Aβ and sRAGE(the soluble, circulating form of RAGE) [34, 35] levelswas also noted, as sRAGE was equally increased in theplasma of AD patients vs. MCI and controls (Fig. 1). Incontrast, parallel measurements of HMGB1 in the MCIcohort was significantly higher than the levels measuredin AD and control subjects. A side-by-side comparisonbetween AD and NL revealed a higher trend for HMGB1

levels in AD patients although the results were not sta-tistically significant. No significant differences betweenthe three groups were observed with respect to S100βlevels.The critical message from these results is that these pro-

inflammatory damage-associated molecules [28] (with theexception of s100β) show specific patterns of expressionin relationship to the continuum from normal aging→M-CI→AD. This strongly suggests that HMGB1 and/orsRAGE in addition to Aβ could have a potential use asprognostic/diagnostic markers for AD and MCI.

Serum expression/activity of soluble thrombomodulin inAD, MCI, and control subjectsAdditionally, in our efforts to develop a validated, non-invasive biomarker for BBB EC damage in AD, wemeasured both sTM antigen (TM-Ag) and TM activity(TMa; which converts protein C to activated PC; see“Methods” section). As shown in Fig. 2, we found signifi-cant increase in TMa above age-matched controls whenMCI and AD levels were grouped (upper panel). We alsoobserved that MCI levels were, in fact, statisticallyhigher than those measured in AD patients (middlepanel). However, as shown in Fig. 2 (bottom panel), thefollowing relationship was found for TM Ag: AD>MCI>-control (lower panel) with a significant increase in ADvs. control subjects. Similar to remitting relapsing mul-tiple sclerosis (RRMS) patients [36], increased levels ofsTM occur in sera of AD and MCI patients as comparedwith controls (Fig. 2).

Effects of proinflammatory HMGB1 and thrombin on BBBpermeability in vitroTo directly assess microvascular damage associated withelevated levels of these proinflammatory DAMPs in ADand MCI patients, we next determined whether HMGB1and/or the proinflammatory coagulation protease, throm-bin, players in vascular barrier dysfunction in other tissuescould disrupt BBB integrity. As shown in Fig. 3a, one-wayANOVA revealed that pre-treatment with recombinanthuman HMGB1 (10 ng/mL) protein for 3 or 6 h signifi-cantly increased BBB apical to basal permeability to FITCdextran across the HBMEC monolayers (p < 0.001 vs. con-trol). We also observed that the magnitude of dextran fluxwas more pronounced with the duration of exposure toHMGB1 (p < 0.01, 6 vs. 3 h; see inset in Fig. 3a). Import-antly, HMGB1-induced increase in BBB paracellular dex-tran permeability could be significantly and specificallyblocked by neutralizing antibody against HMGB1 [37],whereas control (pre-immune) antibody failed to suppressHMGB-1-induced BBB disruption at either incubationtime (Fig. 3a). Intriguingly, our preliminary data also indi-cated that a specific fragment of rTM, called TM-D1, theC-type lectin-like domain [38], can effectively block

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rHMGB1 activity, thus protecting the BBB against thisDAMP (data not shown).Similarly, when we tested the effects of thrombin on BBB

integrity, we observed a significant and concentration-dependent increase in FITC-dextran permeability acrossthe HBMEC monolayers (Fig. 3b). For instance, higherthrombin concentrations (5 nM) more potently inducedBBB disruption (p < 0.0001 vs. control) when compared todextran permeability resulting from exposure to 0.5 or1 nM (p < 0.001). However, we did not observe a furtherelevation in thrombin-induced BBB dextran flux with lon-ger exposure time (3 vs. 6 h), suggesting a maximum andsustained response following 3 h of exposure. The negativeimpact of HMGB1 and thrombin on BBB integrity isfurther confirmed by corresponding immunostaining of

HBMECs that demonstrated a significant downregulationof ZO-1 expression at intercellular tight junctions (TJs)(Fig. 3c).We next determined the extent of BBB disruption

following exposure to HMGB1 by assessing the perme-ability to florescent dextrans of increasing molecularsizes (4–70 kDa) across the hCMEC/D3 monolayers (see“Methods” section). As shown in Fig. 4, HMGB1 expos-ure for 3 h significantly and dose-dependently increasedthe luminal to abluminal flux of all labeled dextransacross the hCMEC/D3 monolayers in a size-selectivemanner. The main effect of the treatment on 4-, 10-,and 70-kDa dextran permeability was significant, asindicated by one-way ANOVA. Further analyses by posthoc tests revealed that high concentrations of HMGB1

Fig. 1 Serum profiling of clinical biomarkers of neuroinflammation to predict the conversion (continuum) of MCI to AD. a The serum levels of Aβ,HMGB1, sRAGE, and s100β are shown individually for each subject belonging to age-matched control (NL), MCI, or AD cohorts to facilitate thecorrelation of these markers. b Serum levels of each DAMP expressed as mean (±SD) for each cohort. *p < 0.05 and ***p < 0.01. n.s. not significant

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(10 and 50 ng/mL) significantly increased apical to basalpermeability of each dextran by 2.0–2.5-fold, as comparedto aged control group (p < 0.05 and p < 0.05, respectively;Fig. 4). Although, HMGB1 at low concentration (5 ng/mL) showed a marked increase in BBB permeability to alldextrans, this effect was non-significant. In addition, weobserved a ceiling effect on BBB permeability to 10 and70 kDa dextrans at HMGB1 (10 ng/ml; Fig. 4), as furtherincrease in concentration to 50 ng/mL did not elevate thepermeability to these dextrans.

DiscussionAlthough rigorous methods are used by the KU ADC toenroll subjects in AD, MCI, MCI due to AD and normalcohorts, one can never fully eliminate other systemic ill-nesses that escaped notice. Such recruitment methodsare part of the approach that our colleagues at the KUADC take, quite similar to methods taken at other insti-tutional ADCs. In this regard, our current results indi-cate that three potent proinflammatory and damage/danger-associated macromolecules are elevated in MCIand AD sera: Aβ, sRAGE, and HMGB1. They furthersuggest that one or more may herald the conversion ofMCI to AD (Fig. 1). Furthermore, our BBB in vitro datashow that one of these, HMGB1, along with the proin-flammatory coagulation protease, thrombin, are proxim-ate mediators of BBB dysfunction. With our presentresults, we conclude that HMGB1 might be a better clin-ical biomarker, along with sRAGE and Aβ, to predict thecourse of neurodegeneration in AD subjects. In contrast,although it also activates RAGE [39], from our results,S100B does not appear to have applicability as a diag-nostic or prognostic marker of neurodegeneration inMCI and AD patients.Considerable evidence indicates that blood levels of

sTM and von Willebrand factor (vWf) can serve assurrogate biomarkers for diffuse microvascular damage[39]. We had previously found that sTM plasma levelswere higher in RRMS patients than in patients withsystemic lupus erythematosus with diffuse microvascularinflammation, indicating its potential utility as a bio-marker for BBB damage [36]. Relevant to our currentresults, others had suggested that sTM, along with vWf,might be a good biomarker for AD-related microvascu-lar damage [40]. In our present studies, soluble forms ofthe anticoagulant/anti-inflammatory TM [38, 41] mightalso serve as a biomarker for microvascular damage ofthe BBB in AD. This is strongly suggested by the patternof expression for the sTM antigen which progressivelyincreases from controls to ↑MCI to ↑AD although onlythe difference between control and AD was statisticallysignificant at this stage. Perhaps future studies involvinga larger cohort of patients will be able to provide add-itional statistical power to the analysis. When data were

Fig. 2 Soluble thrombomodulin activity (TMa) and expression (TM-Ag)in AD, MCI, and control subject sera. TMa and TM-Ag were assayed bykits as described in the “Methods” section. Top panel represents thecombined TMa in MCI and AD patients above age-matched controls.Middle panel shows the differences in TMa between MCI and ADpatients. Bottom panel represents the serum levels of TM-Ag in control,MCI, and AD subjects. Data were expressed as mean ± SEM and *p< 0.05

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Fig. 4 HMGB1 increases BBB permeability to dextrans of various sizes. HCMEC/D3 cells in the transwells were incubated with HMGB1 (5, 10, or50 ng/ml) for 3 h and the apical to basal flux of a mixture of florescent-labeled dextrans of different sizes (FITC-4 kDa; Cascade Blue 10 kDa; andRITC-70 kDa) was assayed following 30 min after the addition of dextrans. (*p < 0.05 vs. control and N = 5/condition)

Fig. 3 HMGB1 and thrombin impair BBB integrity. HBMEC monolayers were incubated with HMGB1 (10 ng/ml) and thrombin (Thr, 0.5, 1, or 5 nM)for 3 or 6 h, and BBB integrity was assessed by paracellular permeability to FITC-dextran. a HMGB1 increases luminal to abluminal dextran fluxacross HBMECs that was reversed by anti-HMGB1 antibody (HMGB1 Ab, 10 ng/ml). The time-dependent increase in HMGB1-induced BBB permeabilitywas shown in the inset. b Exposure to thrombin (3 or 6 h) dose-dependently increases BBB permeability to dextran in HBMECs. c HMGB1 andthrombin disrupt ZO-1 linearity at cell-cell junctions. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control; ●●●p < 0.01 vs. HMGB1 or HMGB1 + controlAb. N = 5/condition

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aggregated, TMa was greater in disease (MCI + AD) thanaged-matched controls (see Fig. 2).As with other vascular barriers [17, 18], human alpha

thrombin significantly compromised BBB integrity invitro at nanomolar concentrations (Fig. 3b), orders ofmagnitude less than the circulating prothrombin levelsin the blood [38]. In an attempt to explain the throm-bin’s effect on brain edema, Guan and colleaguesinjected thrombin stereotactically into the rat caudatenuclei and found a marked extravasation of Evans Bluedye, suggesting BBB disruption [42]. These authors alsodemonstrated that exposure to thrombin upregulatedthe endothelial expression of matrix metalloproteinase-2 possibly through the activation of protease-activatedreceptor 1 (PAR1). In similar direct experiments usingdifferent vascular barrier models, Garcia’s group andothers showed that thrombin mediated barrier disrup-tion, indeed, via a PAR1 mechanism [17, 18, 43]. Al-though, we have no current direct evidence that PAR1mediated the BBB disruption effects of thrombin, thenanomolar concentrations are in agreement with medi-ation via PAR1. However, our preliminary experimentssuggested that the BBB disruptive effects of thrombinare a direct measure of its proteolytic activity, sincerecombinant hirudin, a specific thrombin inhibitor,inhibited the effects of thrombin (data not shown). Fur-ther indicating thrombin was likely interacting with aprotease-activated receptor (PAR) on the EC surface, anrTM fragment that binds both thrombin and PC(TMD23) also blocked thrombin’s effects on BBB per-meability (not shown).The proinflammatory DAMP, HMGB1, dramatically

enhanced BBB permeability to various molecular weightdextrans in primary HBMEC or hCMEC/D3 cultures,following incubation for 3 or 6 h. These BBB changes in-cluding the significant downregulation of ZO-1 expres-sion at intercellular (TJs) clearly mirror what has beenobserved in vivo in animal studies that suggest the pres-ence of amyloid beta is a factor in the “breach” of theBBB [44]. In experimental animals and cell cultures, betaamyloid proteins induce matrix metalloproteinase-9(MMP-9) [45]. Transgenic mutant mice with mutant hu-man amyloid protein show disruption of the BBB [46].Furthermore, transgenic mice with apoE4 demonstrateBBB breakdown by activating the proinflammatorycyclophilin A-MMP-9 pathway in the brain pericytes,which, in turn, results in degradation of the BBB TJs andbasal lamina (BL) proteins [47]. Mice overexpressingAβ-precursor protein have pericyte loss, elevated brainAβ40 and Aβ42 levels accelerating amyloid angiopathy,and cerebral amyloidosis [48].Others have also shown that BBB dysfunction in rats,

whether due to experimental stroke or traumatic braininjury (TBI), can be prevented by a neutralizing antibody

to HMGB1 [49, 50]. Outside the cell, oxidized HMGB1is known to ligate three different pattern recognitionreceptors all expressed on the surface of these ECs (asillustrated in Fig. 5). These include TLR2, TLR4, andRAGE [51, 52], and each can bind other ligands, such asAβ, contributing to vascular complications of other dis-eases [53]. Both TLR and RAGE signaling leads to down-stream NFkB activation with subsequent increase in theexpression/release of tumor necrosis factor (TNF), thusensuring that the inflammatory signal is maintained andamplified [54].Over the last 15 years, the concept that the BBB might

function both as source and target of proinflammatoryfactors is supported by numerous studies showing thatthe cerebral microcirculation is in an “activated proin-flammatory” stage in neurodegenerative diseases such asAD [15, 55] and is a target for various proinflammatoryfactors. PAUSE Circulating Aβ is a proinflammatorycytokine-induced DAMP that binds to RAGE and TLR2[56]. Like S100β and HMGB1, Aβ can exacerbate proin-flammatory cytokine production such as TNF andinterleukin-1 beta (IL-1β); however, in innate immunity,TNF dominates. When HMGB1 binds to RAGE and/orTLRs on microvascular ECs, it releases TNF and othercytokines [37], making the BBB also a source of theseproinflammatory factors, and the axis in AD hasattracted considerable attention [56]. Soluble RAGE orsRAGE, sometimes referred to as endogenous secretoryRAGE, which can be produced either by splicing or pro-teolytic shedding, appears to play the role of a “decoy”to tie up RAGE ligands, such as S100/calgranulin familymembers, HMGB1, or Aβ [39]. The S100B/RAGE axishas been of considerable interest in AD, but earlier re-ports suggested that lower sRAGE levels characterizedAD [39]. Interestingly, we found significant correlationbetween Aβ and HMGB1 or sRAGE (Fig. 1). Further-more, the levels of HMGB1 in serum were in the rangeof rHMGB1 used in the BBB permeability experiments(Figs. 3a and 4), suggesting a continuum from normalthrough MCI to AD may exist. We did not find an indi-cation that S100B might be useful, although a trend ofsignificance in the MCI due to AD cohort was found(Fig. 1).Just in the last decade, it has become increasingly ap-

preciated that inflammation and coagulation are linkedevolutionary defense systems [41], a fact that is slowlybecoming recognized in the CNS as well. TBI and ische-mic and hemorrhagic stroke are all characterized byincreased levels of intraparenchymal thrombin andHMGB1 as well as evidence of BBB dysfunction [42, 49,50]. In the brain, low concentrations of thrombin actthrough its principal receptor, PAR1, to induce neuro-protection [57]. In contrast, at higher concentrations,thrombin causes brain damage [58], where we showed

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Fig. 5 (See legend on next page.)

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that it appears to act via PAR4 [59–61]. Through PARactivation, thrombin directly affects the activity of mul-tiple cell types and regulates a variety of biological func-tions, including inflammation, leukocyte migration, andvascular permeability [61]. Relevant to this and ourcurrent results, all PARs are expressed in microvascularECs [62], and PAR1, at least, is expressed in ECs of theBBB [63].Also attesting to evolutionary linkage, direct relation-

ships also exist between thrombin and HMGB1. HMGB1is involved in a number of systemic vascular diseases[64], is increased in stroke [65], and found around amyloidplaques and NFTs in AD brains [66], where thrombin andprothrombin accumulate as well [67]. Both HMGB1 andthrombin are released in various neurologic conditions,and HMGB1 promotes coagulation [68]. Taken together,these observations raise the possibility that HMGB1 andthrombin also participate during innate immune neuroin-flammatory situations such as in AD and which contributeto BBB dysfunction and WBC transendothelial migration.Finally, we have envisioned our present data in corrob-

oration with previous findings schematically in Fig. 5.This shows that increased levels of danger/damage-asso-ciated, proinflammatory molecules (the alarmin DAMPs,HMGB1 and S100B, as well as Aβ) feature prominentlyin the AD continuum from normal aging through MCIby acting on TLRs and RAGE to induce neuroinflamma-tory signaling at the BBB endothelium. This results inincreased expression of the transcription factor NFkBcausing increased TNF and IL-1β (other interleukins),ROS, and adhesion molecules along with degradation oftight junction (TJ) proteins such as ZO-1. Both endogen-ous as well as exogenous means exist to combat this im-balance including the proteolytic release of sRAGE andsTM, both of which selectively bind up HMGB1 (possiblyother alarmins) preventing engagement of RAGE or TLRs.This also allows for the development of novel therapeutictargets that act like sRAGE and sTM, in a manner lessburdensome and potentially with fewer side effects thanby administering neutralizing antibodies to HMGB1, suchas recently used by others in rat models of stroke, TBI,and Parkinson’s disease [49, 50, 69].

ConclusionsAn increased understanding of the role of HMGB1 andthrombin in BBB dysfunction as well as activation andtransendothelial migration of WBCs contributing toneuroinflammation in AD may allow for the discovery of

novel therapeutic targets and treatment strategies. Notonly might these facilitate treatment to halt progression inAD but these might also aid in means to detect the con-version from MCI to pre-clinical AD, as well as in otherneurological disorders that display BBB dysfunction.

AcknowledgementsWe thank Dr. KY Kim (KUMC) for the preliminary in vitro BBB experimentsand Dr. Michael Bustin (NIH) for the anti-HMGB1 antibody.

FundingThis work was supported by NIH/NIDA R01-DA029121-01A1 and the AlternativeResearch Development Foundation grants received by LC. Some studies werepartially funded by the Kansas Bioscience Authority (BWF). The KU ADC humanstudy was supported by NIH P30 AG035982 to SRH.

Availability of data and materialsAll raw data used in this manuscript will be made available upon request.

Authors’ contributionsBWF contributed to the concept, experimental design, conductance, dataanalysis, and interpretation including manuscript drafting, revision, andapproval; RKS contributed to the conductance, data collection, and analysisin addition to drafting and revising the manuscript; PVD contributed towardthe experimental design, execution, and data analysis; LC contributed toexperimental design, data analysis, manuscript writing and revision, as wellas supporting this study. All authors read and approved the final manuscript.

Competing interestsBWF is the founder and president of pHLOGISTIX LLC.; PVD is the head ofthe Clinical Research Department at Diagnostica Stago, France; RS and LChave no competing interests.

Consent for publicationNot applicable.

Ethics approval and consent to participateEnrollment into the KU ADC clinical cohort was performed using a protocoland consent form/process that was approved by the University of KansasMedical Center’s (KUMC) Human Subject’s Committee (protocol # 11132).

Author details1pHLOGISTIX LLC, 4220 Shawnee Mission Parkway, Fairway, KS 66205, USA.2Department of Neurology, University of Kansas Medical Center, 3901Rainbow Blvd, Kansas City, KS 66160, USA. 3Department of PharmaceuticalSciences, Texas Tech University Health Sciences Center, 1300 S. CoulterStreet, Amarillo, TX 79106, USA. 4Clinical Research Department, R&D,Diagnostica Stago, Gennevilliers, France.

Received: 1 April 2016 Accepted: 17 August 2016

References1. Cummings JL, Morstorf T, Zhong K. Alzheimer's disease drug-development

pipeline: few candidates, frequent failures. Alzheimers Res Ther. 2014;6:37.2. Bloom GS. Amyloid-beta and tau: the trigger and bullet in Alzheimer

disease pathogenesis. JAMA Neurol. 2014;71:505–8.3. Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F,

Cole G. Correlative memory deficits, Abeta elevation, and amyloid plaquesin transgenic mice. Science. 1996;274:99–102.

(See figure on previous page.)Fig. 5 Schematic illustration of proinflammatory Aβ, DAMPs (HMGB1/S100B), and sRAGE acting at the BBB endothelium. Representation of BBB assource and target of neuroinflammation in AD. Endogenous damage molecules, DAMPs as well as Aβ, activate respective receptors to ramp upneuroinflammation. Soluble receptors such as sRAGE and sTM can neutralize some of these, and some may function as distinct biomarkers inprogression of neurodegeneration in AD

Festoff et al. Journal of Neuroinflammation (2016) 13:194 Page 10 of 12

4. Deane R, Bell RD, Sagare A, Zlokovic BV. Clearance of amyloid-beta peptideacross the blood-brain barrier: implication for therapies in Alzheimer'sdisease. CNS Neurol Disord: Drug Targets. 2009;8:16–30.

5. Park L, Zhou P, Koizumi K, El Jamal S, Previti ML, Van Nostrand WE,Carlson G, Iadecola C. Brain and circulating levels of Abeta1-40 differentiallycontribute to vasomotor dysfunction in the mouse brain. Stroke.2013;44:198–204.

6. Tuppo EE, Arias HR. The role of inflammation in Alzheimer's disease.Int J Biochem Cell Biol. 2005;37:289–305.

7. Hensley K. Neuroinflammation in Alzheimer's disease: mechanisms,pathologic consequences, and potential for therapeutic manipulation.J Alzheimers Dis. 2010;21:1–14.

8. Mhatre M, Floyd RA, Hensley K. Oxidative stress and neuroinflammation inAlzheimer's disease and amyotrophic lateral sclerosis: common links andpotential therapeutic targets. J Alzheimers Dis. 2004;6:147–57.

9. Ferretti MT, Cuello AC. Does a pro-inflammatory process precedeAlzheimer's disease and mild cognitive impairment? Curr Alzheimer Res.2011;8:164–74.

10. Zlokovic BV. Neurovascular pathways to neurodegeneration in Alzheimer'sdisease and other disorders. Nat Rev Neurosci. 2011;12:723–38.

11. Muoio V, Persson PB, Sendeski MM. The neurovascular unit—conceptreview. Acta Physiol (Oxf). 2014;210:790–8.

12. Keaney J, Campbell M. The dynamic blood-brain barrier. FEBS J.2015;282:4067–79.

13. Redwine L, Mills PJ, Sada M, Dimsdale J, Patterson T, Grant I. Differentialimmune cell chemotaxis responses to acute psychological stress inAlzheimer caregivers compared to non-caregiver controls. Psychosom Med.2004;66:770–5.

14. Zaghi J, Goldenson B, Inayathullah M, Lossinsky AS, Masoumi A, Avagyan H,Mahanian M, Bernas M, Weinand M, Rosenthal MJ, et al. Alzheimer diseasemacrophages shuttle amyloid-beta from neurons to vessels, contributing toamyloid angiopathy. Acta Neuropathol. 2009;117:111–24.

15. Grammas P, Martinez J, Miller B. Cerebral microvascular endothelium and thepathogenesis of neurodegenerative diseases. Expert Rev Mol Med. 2011;13:e19.

16. Heppner FL, Ransohoff RM, Becher B. Immune attack: the role ofinflammation in Alzheimer disease. Nat Rev Neurosci. 2015;16:358–72.

17. Birukova AA, Birukov KG, Smurova K, Adyshev D, Kaibuchi K, Alieva I, GarciaJG, Verin AD. Novel role of microtubules in thrombin-induced endothelialbarrier dysfunction. FASEB J. 2004;18:1879–90.

18. Garcia JG. Concepts in microvascular endothelial barrier regulation in healthand disease. Microvasc Res. 2009;77:1–3.

19. Wolfson RK, Chiang ET, Garcia JG. HMGB1 induces human lung endothelialcell cytoskeletal rearrangement and barrier disruption. Microvasc Res.2011;81:189–97.

20. Nawaz MI, Mohammad G. Role of high-mobility group box-1 protein indisruption of vascular barriers and regulation of leukocyte-endothelialinteractions. J Recept Signal Transduct Res. 2015;35:340–5.

21. Dumitriu IE, Baruah P, Manfredi AA, Bianchi ME, Rovere-Querini P. HMGB1:guiding immunity from within. Trends Immunol. 2005;26:381–7.

22. Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M, Che J, FrazierA, Yang H, Ivanova S, Borovikova L, et al. HMG-1 as a late mediator ofendotoxin lethality in mice. Science. 1999;285:248–51.

23. Schiraldi M, Raucci A, Munoz LM, Livoti E, Celona B, Venereau E, Apuzzo T,De Marchis F, Pedotti M, Bachi A, et al. HMGB1 promotes recruitment ofinflammatory cells to damaged tissues by forming a complex with CXCL12and signaling via CXCR4. J Exp Med. 2012;209:551–63.

24. Venereau E, Schiraldi M, Uguccioni M, Bianchi ME. HMGB1 and leukocytemigration during trauma and sterile inflammation. Mol Immunol.2013;55:76–82.

25. Mazarati A, Maroso M, Iori V, Vezzani A, Carli M. High-mobility group box-1impairs memory in mice through both toll-like receptor 4 and receptor foradvanced glycation end products. Exp Neurol. 2011;232:143–8.

26. Iwashyna TJ, Ely EW, Smith DM, Langa KM. Long-term cognitive impairmentand functional disability among survivors of severe sepsis. JAMA. 2010;304:1787–94.

27. Frank-Cannon TC, Alto LT, McAlpine FE, Tansey MG. Doesneuroinflammation fan the flame in neurodegenerative diseases? MolNeurodegener. 2009;4:47.

28. Clark IA, Vissel B. Amyloid beta: one of three danger-associated moleculesthat are secondary inducers of the proinflammatory cytokines that mediateAlzheimer's disease. Br J Pharmacol. 2015;172:3714–27.

29. McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack Jr CR, Kawas CH,Klunk WE, Koroshetz WJ, Manly JJ, Mayeux R, et al. The diagnosis ofdementia due to Alzheimer's disease: recommendations from the NationalInstitute on Aging-Alzheimer's Association workgroups on diagnosticguidelines for Alzheimer's disease. Alzheimers Dement. 2011;7:263–9.

30. Albert MS, DeKosky ST, Dickson D, Dubois B, Feldman HH, Fox NC, Gamst A,Holtzman DM, Jagust WJ, Petersen RC, et al. The diagnosis of mild cognitiveimpairment due to Alzheimer's disease: recommendations from theNational Institute on Aging-Alzheimer's Association workgroups ondiagnostic guidelines for Alzheimer's disease. Alzheimers Dement.2011;7:270–9.

31. Sajja RK, Green KN, Cucullo L. Altered Nrf2 signaling mediateshypoglycemia-induced blood-brain barrier endothelial dysfunction in vitro.PLoS One. 2015;10:e0122358.

32. Dhillon NK, Peng F, Bokhari S, Callen S, Shin SH, Zhu X, Kim KJ, Buch SJ.Cocaine-mediated alteration in tight junction protein expression andmodulation of CCL2/CCR2 axis across the blood-brain barrier: implicationsfor HIV-dementia. J Neuroimmune Pharmacol. 2008;3:52–6.

33. Van Dreden P, Rousseau A, Savoure A, Lenormand B, Fontaine S, Vasse M.Plasma thrombomodulin activity, tissue factor activity and high levels ofcirculating procoagulant phospholipid as prognostic factors for acutemyocardial infarction. Blood Coagul Fibrinolysis. 2009;20:635–41.

34. Santilli F, Vazzana N, Bucciarelli LG, Davi G. Soluble forms of RAGE in humandiseases: clinical and therapeutical implications. Curr Med Chem. 2009;16:940–52.

35. Yamagishi S, Matsui T. Soluble form of a receptor for advanced glycationend products (sRAGE) as a biomarker. Front Biosci (Elite Ed). 2010;2:1184–95.

36. Festoff BW, Li C, Woodhams B, Lynch S. Soluble thrombomodulin levels inplasma of multiple sclerosis patients and their implication. J Neurol Sci.2012;323:61–5.

37. Fiuza C, Bustin M, Talwar S, Tropea M, Gerstenberger E, Shelhamer JH,Suffredini AF. Inflammation-promoting activity of HMGB1 on humanmicrovascular endothelial cells. Blood. 2003;101:2652–60.

38. Conway EM. Thrombomodulin and its role in inflammation. SeminImmunopathol. 2012;34:107–25.

39. Leclerc E, Sturchler E, Vetter SW. The S100B/RAGE axis in Alzheimer'sdisease. Cardiovasc Psychiatry Neurol. 2010;2010:539581.

40. Yavuz BB, Dede DS, Yavuz B, Cankurtaran M, Halil M, Ulger Z, CankurtaranES, Aytemir K, Kabakci G, Haznedaroglu IC, Ariogul S. Potential biomarkersfor vascular damage in Alzheimer's disease: thrombomodulin and vonWillebrand factor. J Nutr Health Aging. 2010;14:439–41.

41. Delvaeye M, Conway EM. Coagulation and innate immune responses: canwe view them separately? Blood. 2009;114:2367–74.

42. Guan JX, Sun SG, Cao XB, Chen ZB, Tong ET. Effect of thrombin onblood brain barrier permeability and its mechanism. Chin Med J (Engl).2004;117:1677–81.

43. Bogatcheva NV, Garcia JG, Verin AD. Molecular mechanisms of thrombin-induced endothelial cell permeability. Biochemistry (Mosc). 2002;67:75–84.

44. Carmeliet P, De Strooper B. Alzheimer's disease: a breach in the blood-brainbarrier. Nature. 2012;485:451–2.

45. Hartz AM, Bauer B, Soldner EL, Wolf A, Boy S, Backhaus R, Mihaljevic I,Bogdahn U, Klunemann HH, Schuierer G, Schlachetzki F. Amyloid-betacontributes to blood-brain barrier leakage in transgenic human amyloidprecursor protein mice and in humans with cerebral amyloid angiopathy.Stroke. 2012;43:514–23.

46. Kook SY, Seok Hong H, Moon M, Mook-Jung I. Disruption of blood-brainbarrier in Alzheimer disease pathogenesis. Tissue Barriers. 2013;1:e23993.

47. Bell RD, Winkler EA, Singh I, Sagare AP, Deane R, Wu Z, Holtzman DM,Betsholtz C, Armulik A, Sallstrom J, et al. Apolipoprotein E controlscerebrovascular integrity via cyclophilin A. Nature. 2012;485:512–6.

48. Sagare AP, Bell RD, Zhao Z, Ma Q, Winkler EA, Ramanathan A, Zlokovic BV.Pericyte loss influences Alzheimer-like neurodegeneration in mice. NatCommun. 2013;4:2932.

49. Zhang J, Takahashi HK, Liu K, Wake H, Liu R, Maruo T, Date I, Yoshino T,Ohtsuka A, Mori S, Nishibori M. Anti-high mobility group box-1 monoclonalantibody protects the blood-brain barrier from ischemia-induced disruptionin rats. Stroke. 2011;42:1420–8.

50. Okuma Y, Liu K, Wake H, Zhang J, Maruo T, Date I, Yoshino T, Ohtsuka A,Otani N, Tomura S, et al. Anti-high mobility group box-1 antibody therapyfor traumatic brain injury. Ann Neurol. 2012;72:373–84.

51. Hori O, Brett J, Slattery T, Cao R, Zhang J, Chen JX, Nagashima M, Lundh ER,Vijay S, Nitecki D, et al. The receptor for advanced glycation end products

Festoff et al. Journal of Neuroinflammation (2016) 13:194 Page 11 of 12

(RAGE) is a cellular binding site for amphoterin. Mediation of neuriteoutgrowth and co-expression of rage and amphoterin in the developingnervous system. J Biol Chem. 1995;270:25752–61.

52. van Beijnum JR, Buurman WA, Griffioen AW. Convergence and amplificationof toll-like receptor (TLR) and receptor for advanced glycation end products(RAGE) signaling pathways via high mobility group B1 (HMGB1).Angiogenesis. 2008;11:91–9.

53. Bierhaus A, Humpert PM, Stern DM, Arnold B, Nawroth PP. Advancedglycation end product receptor-mediated cellular dysfunction. Ann N YAcad Sci. 2005;1043:676–80.

54. Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B, SternDM, Nawroth PP. Understanding RAGE, the receptor for advanced glycationend products. J Mol Med (Berl). 2005;83:876–86.

55. Rochfort KD, Cummins PM. The blood-brain barrier endothelium: a targetfor pro-inflammatory cytokines. Biochem Soc Trans. 2015;43:702–6.

56. Liu S, Liu Y, Hao W, Wolf L, Kiliaan AJ, Penke B, Rube CE, Walter J, HenekaMT, Hartmann T, et al. TLR2 is a primary receptor for Alzheimer's amyloidbeta peptide to trigger neuroinflammatory activation. J Immunol.2012;188:1098–107.

57. Striggow F, Riek M, Breder J, Henrich-Noack P, Reymann KG, Reiser G.The protease thrombin is an endogenous mediator of hippocampalneuroprotection against ischemia at low concentrations but causesdegeneration at high concentrations. Proc Natl Acad Sci U S A.2000;97:2264–9.

58. Xi G, Reiser G, Keep RF. The role of thrombin and thrombin receptors inischemic, hemorrhagic and traumatic brain injury: deleterious or protective?J Neurochem. 2003;84:3–9.

59. Suo Z, Wu M, Citron BA, Gao C, Festoff BW. Persistent protease-activatedreceptor 4 signaling mediates thrombin-induced microglial activation. J BiolChem. 2003;278:31177–83.

60. Suo Z, Wu M, Citron BA, Palazzo RE, Festoff BW. Rapid tau aggregation anddelayed hippocampal neuronal death induced by persistent thrombinsignaling. J Biol Chem. 2003;278:37681–9.

61. Suo Z, Citron BA, Festoff BW. Thrombin: a potential proinflammatorymediator in neurotrauma and neurodegenerative disorders. Curr DrugTargets Inflamm Allergy. 2004;3:105–14.

62. Hirano K. The roles of proteinase-activated receptors in the vascularphysiology and pathophysiology. Arterioscler, Thromb, Vasc Biol.2007;27:27–36.

63. Bartha K, Domotor E, Lanza F, Adam-Vizi V, Machovich R. Identification ofthrombin receptors in rat brain capillary endothelial cells. J Cereb BloodFlow Metab. 2000;20:175–82.

64. de Souza AW, Westra J, Limburg PC, Bijl M, Kallenberg CG. HMGB1 invascular diseases: its role in vascular inflammation and atherosclerosis.Autoimmun Rev. 2012;11:909–17.

65. Fang P, Schachner M, Shen YQ. HMGB1 in development and diseases of thecentral nervous system. Mol Neurobiol. 2012;45:499–506.

66. Takata K, Takada T, Ito A, Asai M, Tawa M, Saito Y, Ashihara E, Tomimoto H,Kitamura Y, Shimohama S. Microglial amyloid-beta1-40 phagocytosisdysfunction is caused by high-mobility group box protein-1: implications forthe pathological progression of Alzheimer's disease. Int J Alzheimers Dis.2012;2012:685739.

67. Arai T, Miklossy J, Klegeris A, Guo JP, McGeer PL. Thrombin andprothrombin are expressed by neurons and glial cells and accumulate inneurofibrillary tangles in Alzheimer disease brain. J Neuropathol Exp Neurol.2006;65:19–25.

68. Ito T, Kawahara K, Nakamura T, Yamada S, Nakamura T, Abeyama K,Hashiguchi T, Maruyama I. High-mobility group box 1 protein promotesdevelopment of microvascular thrombosis in rats. J Thromb Haemost.2007;5:109–16.

69. Sasaki T, Liu K, Agari T, Yasuhara T, Morimoto J, Okazaki M, Takeuchi H,Toyoshima A, Sasada S, Shinko A, et al. Anti-high mobility group box 1antibody exerts neuroprotection in a rat model of Parkinson's disease.Exp Neurol. 2016;275(Pt 1):220–31.

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